Journal of Young Pharmacists 5 (2013) 7e12

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Journal of Young Pharmacists

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Original article

Anti-diabetic activities of (part) and fractions of Stereospermum tetragonum DC

Renjit Bino Kingsley a, Manisha Mishra b, Pemaiah Brindha b, Appian Subramoniam a, *
a
Division of Phytochemistry and Phytopharmacology, Tropical Botanic Garden and Research Institute, Palode, Thiruvananthapuram 695562, India
b
Department of Ayurvedic Research, Sastra University, Tanjore, India

a r t i c l e i n f o a b s t r a c t

Article history: Objectives: To identify the active principles, determine the anti-diabetes activity of fraction of
Received 11 April 2012 Stereospermum tetragonum root.
Accepted 1 September 2012 Materials and Methods: The efficacy was evaluated in streptozotocin induced type 2 diabetic rats and the
Available online xxx
anti-hyperglycemic activity was studied by glucose tolerance test. The major active compounds were
isolated by solvent fractionation and chromatographic techniques and characterized with spectral data.
Keywords:
Results: The active fraction of S. tetragonum showed presence of anti-diabetes mellitus activity in type-2
Iridoid glycoside
diabetic rats. It did not significantly influence insulin release from cultured islets. Two active principles
Naphthoquinone derivative
Stereospermum tetragonum
(active at 2 mg/kg dose) were isolated and characterized with spectral data. One of them was identified as an
Anti-diabetes mellitus iridoid type glycoside and the other one was a lapachol like compound (derivative of naphthoquinone).
Conclusions: Two active principles from the anti-diabetes fraction of S. tetragonum root were isolated and
identified as an iridoid glycoside and a naphthoquinone derivative.
Copyright Ó 2013, InPharm Association, Published by Reed Elsevier India Pvt. Ltd. All rights reserved.

1. Introduction against streptozotocin-induced type-2 DM and to identify its active

Diabetes mellitus (DM) is a major disease affecting nearly 10% of
the adult population of the world. In the recent past, many hy-
poglycemic agents are introduced; still DM and its complications
continue to be a major medical problem.1 There are many plants
used in traditional medicine to treat DM, but almost all of these
plants are not widely acceptable and satisfactory for treating DM.
Many more plants are used in local health traditions, especially in
rural areas, to treat DM. These plants are not known to the
mainstream population.2,3 So there is an urgent need to search and
find out such plants and to determine their utility to develop
valuable therapeutic agents with modern standards of safety and
efficacy. In this connection, previous studies carried out in this
laboratory using experimental animal models have shown anti-DM
activity of Artemisia pallen,4 Cassia kleinii,5 and Hemionitis arifolia.6
Recent preliminary studies have showed promising anti-hyper-
glycemic activity of the roots of Stereospermum tetragonum DC.
(family: Bignoniaceae) in fed, not fasted, rats.7 This plant root is
used in folk medicine to treat DM in certain remote villages of
Tamil Nadu, India. Hence the present study was carried out to
determine the efficacy of the active fraction of S. tetragonum
principles.
2. Materials and methods
2.1. Collection of plant materials
S. tetragonum root (family: Bignoniaceae) was collected from
Tirunelveli and Chengalpatu districts of Tamil Nadu, India and
identified by the taxonomist of TBGRI and a voucher specimen
(TBGRI 8282) has been deposited in the institute herbarium.
2.2. Animals
Inbred Wister rats (150e200 g weight), reared in TBGRI animal
house were used. Animals were caged in uniform hygienic
conditions and fed with standard pellet diet (Lipton Indian Ltd,
Bangalore) and water ad libitum as per the guide lines of Institute
Animal Ethics Committee (IAEC). IAEC is controlled by Committee
for the Purpose of Control and Supervision of Experiments on
Animals [Approval No: B-31/03/2010/PPD-7 dated 31-03-2010].
2.3. Isolation of an active fraction (AF)

* Corresponding author. T.C. 14/80, Near KIMS, Anayara, Thiruvananthapuram

695029, India. Tel.: þ91 (0) 471 2443609, þ91 09496093609 (mobile); fax: þ91 (0)
The water extract of S. tetragonum root powder was precipitated
472 2869646. with absolute ethanol (1:1 v/v) and separated in to precipitate and
E-mail address: asubramoniam@yahoo.com (A. Subramoniam). soluble fractions. The activity was found in the soluble fraction.7

0975-1483/$ e see front matter Copyright Ó 2013, InPharm Association, Published by Reed Elsevier India Pvt. Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.jyp.2012.09.002
8 R. Bino Kingsley et al. / Journal of Young Pharmacists 5 (2013) 7e12
This active fraction (AF) was used for efficacy evaluation against
streptozotocin-induced type-2 diabetic rats and mechanism of ac-
tion studies.
2.4. Determination of the efficacy of AF
Streptozotocin-induced type-2 diabetic rats were produced by
injecting 80 mg/kg streptozotocin (i.p.) in citrate buffer on the 5th
day of their birth and this model is called neonatal-5 STZ induced
rat model of type-2 DM.8 Eight weeks later, blood samples were
drawn and glucose levels were determined to confirm induction of
diabetes. The diabetic rats which showed blood glucose levels in
the range of 11.5e12.0 mmol/L were selected for efficacy evaluation
of the herbal drug.9
To evaluate the efficacy, the streptozotocin diabetic rats were
divided into three groups of six each. The diabetic control group
was given 1.5 mL of water, p.o., daily. The test group was given daily
dose of AF (25 mg/kg). This dose was found to be the optimum dose
in our glucose tolerance test (GTT). The third group received gli-
benclamide (0.5 mg/kg). Weight and sex matched six normal rats
were kept as control group (4th group). Treatment was continued
for 21 days. Blood samples were collected on days 1, 7, 14 and 21. On
day 21, animals were killed after blood collection and liver samples
were removed for glycogen estimation.6
2.5. Estimation of serum biochemical parameters
Serum glucose was estimated spectrophotometerically using a
commercial assay kit (Monozyme, India ltd). Liver glycogen was
estimated as described.6 Lipid peroxides in blood serum of strep-
tozotocin diabetic rats was estimated as per Okhawa et al.10
Reduced glutathione in the serum was estimated as per standard
method.11 Vitamin C in the serum was estimated as described.12
2.6. Glucose tolerance test (GTT)
Normal fed rats were divided into indicated number of groups.
Control group received the vehicle. The experimental groups received
indicated doses of AF, sub-fractions or isolated compounds in an
identical manner. Thirty minutes after herbal drug or vehicle (control)
administration, the rats of all the groups were loaded with 60% glucose
(3 g/kg, p.o.). Blood samples were collected by retro-orbital puncture
under anesthesia,13 just 1 min prior to herbal drug administration, and
at 30 and 90 min after glucose loading. Serum glucose levels were
measured immediately. Six fed animals were used in each group.7
2.7. Determination of the effect of AF on the release of insulin from
rat pancreatic b-cells
Isolation of rat pancreatic b-cell islets and insulin release assay
with or without the AF were carried out as per the method of
Bhonde et al.14
The islets cultured for 48 h in RPMI-1640 medium supple-
mented with 10% fetal calf serum were checked for viability by
Trypan blue exclusion method.
2.7.1. Insulin release assay
Ten cultured viable b-cell islets were placed in each of the well in a
24 well plate in such a manner to get triplicate of six groups. All these
wells contained 1 mL of KRBH (pH-7.4), supplemented with
2.75 mmol/L of glucose. The islets were pre-incubated for 1 h at 37  C
in a CO2 incubator, gassed with 5% CO2 in air and supernatants were
separated. After pre-incubation, the six groups were incubated again
for 1 h with 1 mL of KRBH with or without glucose and S. tetragonum
(AF). The first group (control) of b-cell islets was incubated in 1 mL of
KRBH. The second group (glucose control group) was incubated in
1 mL of KRBH with 16.5 mmol/L glucose. Third and fourth groups of
islets were incubated in 1 mL of KRBH and 10 and 40 mg/mL AF
respectively. Fifth and sixth groups were incubated as third and fourth
groups but with 16.5 mmol/L glucose. After incubation, the islets were
spun down and supernatants were collected. The insulin released in
the supernatant was measured by radio-immuno-assay method.15
2.8. Isolation of active principles
2.8.1. Separation of AF into 3 sub-fractions
The AF was subjected to silica gel-G (E. Merck Ltd. Mumbai)
preparative thin layer chromatography (TLC) using butanol:acetic
acid:water (8:2:2 v/v) as solvent system. [When the chromato-
grams were sprayed with ansaldehyde sulfuric acid reagent and
heated at 110  C for 5 min, the AF was resolved into 10 compo-
nents]. The components separated on large scale preparative TLC
plates were grouped into 3 sub-fractions namely sub-fraction-1
(bottom 3 bands/components), sub-fraction-2 (middle 4 bands),
and sub-fraction-3 (top 3 bands). These three groups of bands were
eluted separately with methanol and filtered free of silica gel and
dried.16 The three sub-fractions were tested for their glucose
lowering activity by GTT in rats. [The first and third fractions
showed significant glucose lowering effect].
2.8.2. Separation of active compounds from sub-fractions 1 and 3
by column chromatography
The sub-fraction-1 (3.2 g) was subjected to column chroma-
tography [50 cm  25 mm] using 70 g silica gel (60e120 mesh size)
and eluting with increasing polarity of solvents. The sub-fraction
was eluted with 100% chloroform [100 mL  5], chlor-
oform:methanol (95:5 v/v) [100 mL  5], chloroform:methanol
(90:10 v/v) [100 mL  5], chloroform:methanol (85:15 v/v)
[100 mL  5], chloroform:methanol (80:20 v/v) [100 mL  5],
chloroform:methanol (75:25) [100 mL  5], chloroform:methanol
(60:40) [100 mL  5], chloroform:methanol (40:60) [100 mL  5],
chloroform:methanol (25:75) [100 mL  5] and methanol (100%).
About 100 mL fractions were collected. The fractions collected were
monitored by silica gel 60 F254 TLC. Similar fractions as judged from
TLC were combined together. A pure compound was obtained in
the fractions of chloroform: methanol (25:75, v/v). HPTLC profile of
the active fraction and isolated compounds was done using CAMAG
HPTLC system equipped with sample applicator LINOMAT- V,
Scanner III and integration software CATS 4.04.
The sub-fraction-3 (2.4 g) was subjected to column chromatog-
raphy [50 cm  25 mm] using 70 g Merck silica gel (60e120 mesh
size) and eluting with increasing polarity of solvents. The eluting
solvents were 100% chloroform [100 mL  5], chloroform:methanol
(95:5 v/v) [100 mL  5], chloroform:methanol (90:10 v/v)
[100 mL  5] and chloroform:methanol (85:15 v/v) [100 mL  5].
About 100 mL fractions were collected. The fractions collected were
monitored by silica gel 60 F254 TLC. Silica gel plates were developed
using butanol:acetic acid:water (8:2:2 v/v) as the mobile phase.
1.7 g of fractions eluted in chloroform:methanol (85:15 v/v)
were again loaded into a packed silica gel (60 g; 60e120 mesh size)
column (75 cm  12.5 cm). The eluting solvents or solvent mixtures
(increasing polarity) were (100%) chloroform [25 mL  5], chlor-
oform:methanol (95:5 v/v) [25 mL  5], and chloroform:methanol
(93:7 v/v) [25 mL  33]. About 25 mL fractions were collected. The
eluted fractions were monitored by silica gel 60 F254 TLC as above.
Similar fractions were combined together, allowed to dry and
weighed.
1.2 g of fractions eluted in chloroform:methanol (93:7 v/v) were
again loaded to a packed silica gel (40 g; 60e120 mesh size) column
(75 cm  12.5 cm). The eluting solvents were 100% chloroform
 R. Bino Kingsley et al. / Journal of Young Pharmacists 5 (2013) 7e12 9

[25 mL  3], chloroform:methanol (98:2 v/v) [10 mL  5], chlor-

oform:methanol (90:10 v/v) [10 mL  5] and chloroform:methanol
(80:20, v/v) [25 mL  10]. About the same volume of solvents as
poured was collected as fractions. The fractions collected were
monitored by silica gel 60 F254 TLC. A pure compound was obtained
in chloroform:methanol (80:20, v/v) fractions.

2.8.3. Characterization of the active principles

All the IR spectra were measured using a Nicolet 170SX. Liquid
samples were measured with liquid film method, and solid samples
were measured by using KBr disc and Nujol paste methods.
1
H NMR was measured with a JEOL AL-400 (399.65 MHz). The
measuring conditions for the most of the spectra were as follows:
flip angle of 22.5e30.0 , pulse repetition time of 30 s. The long
pulse repetition time and small flip angle were used to ensure
precise relative intensities. The 1H NMR chemical shifts were
referred to TMS in organic solvents and TSP in D2O.
13
C NMR was measured with a Bruker AC-200 (50.323 MHz). The
measuring conditions for the most of the spectra were as follows: a
pulse flips angle of 22.5e45 , a pulse repetition time of 4e7 s, and a
Fig. 1. Effect of active fraction of S. tetragonum on the levels of glucose in streptozo-
resolution of 0.025e0.045 ppm. The spectra whose spectral codes
tocin-induced diabetic rats. Values are mean  S.D.; n ¼ 6, *P < 0.05, **P < 0.001
started with “CDS” were reconstructed from peak positions, in- (compared to diabetic control).
tensities and line widths by assuming all resonance peaks were
Lorenz lines. The chemical shift was referred to a TMS for all solvents.
Measurements were performed by LC-MS with ESI probe. The period. The effect was almost comparable to that of 0.5 mg/kg
isolated compound was injected into an end capped C18 reverse glibenclamide.
phase column (250 mm  4.0 mm, 5 mm) [E. Merck] and eluted at a The body weight loss and drastic reduction in liver glycogen
flow rate of 0.5 mL/min in isocratic mode with a mobile phase observed in the diabetic animals were restored, to a large extent, by
consisted of methanol and water (90:10 v/v) by keeping MS scan the herbal drug administration (Table 1). AF (25 mg/kg) adminis-
range 50e850 AMU. The mass peaks were observed respective to tration restored the altered levels of serum biochemical parameters
their m/z values. Drying gas flow was adjusted at 10 L/h and of diabetic animals, almost to the normal levels seen in normal
temperature was fixed at 250  C. control rats. The serum cholesterol and triglyceride levels were
substantially improved in AF treated diabetic animals. Vitamin C
and glutathione levels were markedly decreased in the diabetic
2.9. Statistical analysis
rats. AF treatment resulted in substantial improvement in the levels
of vitamin C and glutathione (Table 1). The elevated levels of serum
Statistical comparisons were done using one-way analysis of
lipid peroxides seen in the diabetic animals were reduced to
variance (ANOVA) followed by Dunnett’s test. P values <0.05 were
marginally below normal levels by AF treatment (Table 1). It is of
considered significant.
interest to note that the drug decreased both oxidative stress and
hyperlipidemic conditions seen in diabetic rats. These conditions
3. Results are responsible to a large extent for the cardiovascular complica-
tions associated with diabetes.
3.1. Effect of AF on streptozotocin-induced type-2 diabetic rats This study reports for the first time the anti-DM (type-2)
activity of the active fraction (AF) isolated from S. tetragonum
As given in Fig. 1, the active fraction from S. tetragonum (AF) root. The anti-DM activity of the active fraction (25 mg/kg) was
showed significant anti-DM activity in streptozotocin-induced almost comparable to that of glibenglamide (500 mg/kg). We
type-2 diabetic rats. The daily drug administration at a dose of have already shown that the active fraction has activity against
25 mg/kg gradually decreased the serum glucose levels from 11.7 to alloxan-induced diabetes in rats.7 Thus, the AF is effective in
8.2 mmol/L in 21 days. In untreated diabetic control rats, the both alloxan-induced DM and streptozotocin-induced type-2
glucose levels increased from 11.9 to 14.6 mmol/L during the same DM.

Table 1
Effect of active fraction from Stereospermum tetragonum on body weight, liver weight, liver glycogen and serum biochemical parameters in streptozotocin-induced diabetic
rats.

Parameters Normal control Diabetic control Diabetic treated with Diabetic treated with
active fraction (25 mg/kg) glibenclamide (0.5 mg/kg)
Final body weight (g)a 227.3  7.8 139.3  9.1 213.7  7.1** 203.3  10.1**
Liver weight (g) 4.94  0.95 6.90  0.18 6.09  0.12* 5.69  0.22**
Liver glycogen (mg/g wet tissue) 11.98  1.46 5.19  0.13 10.74  0.15** 11.09  0.22**
Cholesterol (mmol/L) 2.50  0.4 3.57  0.04 2.96  0.05* 2.69  0.04**
Triglycerides (mmol/L) 6.10  0.19 17.35  0.17 9.57  0.22** 9.58  0.17**
Lipid peroxides (mmol/L) 4.28  0.32 5.91  0. 33 3.85  0.20* 4.05  0.19*
Glutathione (mmol/L) 0.91  0.05 0.56  0.04 0.79  0.03* 0.82  0.04*
Vitamin C (mmol/L) 128.1  10.0 45.1  2.5 101.0  6.3** 106.3  3.5**

Values are mean  S.D.; n ¼ 6; *P < 0.05, **P < 0.001 (compared to diabetic control) [One-way ANOVA followed by Dunnett’s post hoc comparison was done].
a
Initial body weight was not significantly different among different groups.
10 R. Bino Kingsley et al. / Journal of Young Pharmacists 5 (2013) 7e12

influence the insulin release at the concentrations (10e40 mg/mL)

studied both in the presence or absence of high level of glucose
(16.5 mmol/L).

3.3. Effect of chemical fractions and principles on glucose tolerance

in glucose loaded normal rats

As shown in Table 2, AF was found to be effective in glucose

tolerance test (GTT) when administered orally as well as intra-peri-
toneally. But, it did not influence substantially the levels of serum
glucose in intra-peritoneally glucose loaded rats, in contrast to oral
glucose administration to fed rats. Thus, it appears that glucose ab-
Fig. 2. Effect of active fraction of S. tetragonum on insulin release from isolated and sorption/transport from the intestinal tract is inhibited by the AF. It
cultured pancreatic b-cell islets. Values are mean  S.D.; n ¼ 3; insulin values in can be assumed that the herbal drug directly or indirectly inhibits, to
glucose added groups are significantly different compared to control, P < 0.001. a large extent, glucose absorption or transport from the intestine.
The AF from S. tetragonum root was resolved into 10 components
on TLC and HPTLC (Fig. 3 A and B). Using large scale preparative silica
Table 2
gel-G TLC, AF was separated in to three sub-fractions (Fig. 3 A) and
Effect of different routes of administration of the active fraction as well as glucose on
glucose tolerance in fed normal rats. the percentage of yield of sub-fraction-1 (SF-1), SF-2 and SF-3 were
about 29, 39, 19.3% respectively of AF. When all these three sub-
Serum glucose levels (mmol/L)
fractions were tested for glucose lowering activity in fed and orally
Treatment Time (min) after glucose administration. glucose loaded normal rats, at the dose of 25 mg/kg body weight
0 (initial) 30 90 each, SF-1 and SF-3 showed significant glucose lowering activity. SF-
Control (oral glucose) 5.31  0.30 7.81  0.50 6.27  0.22 2 did not show any reduction in serum glucose levels (Table 3).
Active fraction 5.49  0.26 6.26  0.8** 5.65  0.33* The major active compounds in SF-1 and SF-3 were isolated by
(25 mg/kg; oral) column chromatography in pure forms (Fig. 3 A and B). The com-
þ oral glucose pound isolated from SF-1 (C1) and SF-3 (C2) showed significant anti-
Active fraction 5.43  0.25 6.19  0.47** 5.76  0.26*
(25 mg/kg; I.P.)
hyperglycemic activity at a relatively low concentration (2 mg/kg
þ oral glucose body weight) in GTT on fed and glucose loaded normal rats (Table 3).
Control (I.P. glucose) 5.30  0.31 7.86  0.52 6.18  0.29
Active fraction 5.28  0.28 7.42  0.59 6.25  0.39
(25 mg/kg; oral) 3.4. Spectral analysis and elucidation of structures of the
þ I.P. glucose compounds
Values are mean  S.D.; n ¼ 6; **, P < 0.001, *, P < 0.05 (compared to respective
control values, Student’s t test); I.P., intraperitoneal. Compound 1 (C1): IR (KBr): 3400 (OeH str.), 2900 (CeH
aliphatic str.), 1000 (CeO str.) cm1. MS: m/z 437 (MD. 432 þ 5)
of C19 H28 O11 1H NMR (CD3OD 400 MHz). d 8.52 (s, 1 H, C4eCOOH),
3.2. Effect of AF on insulin release from cultured pancreatic b-cell islets 5.169 (s, 1 H, C3eH), 5.162 (s, 1H, C6eH), 5.14 (d, 1H,C1eH), 4.70
(s, 1H, C0 2eH) 4.68 (brd.1H, C7aeH), 4.55 (t, 1H, C0 3eH), 4.14 (s, 1H,
As shown in Fig. 2, glucose markedly stimulated insulin release C0 6eCH2eOH), 4.12 (s, 1H, C7eCH2eOH) 4.08e4.03 (m, 3H, C0 45&6e
into the medium from cultured pancreatic b-cell islets. AF did not H), 3.95 (s, 1H, C5eOH) 3.79 (s, 3H, C0 3eOCH3), 3.78 (s, 3H, C0 4e

Fig. 3. Thin layer chromatographic separation of active fraction (AF) and isolated compounds from S. tetragonum root. A. TLC separation of AF from the water extract of S. tetragonum
using silica gel 60 F254 and the solvent system, butanol:aceticacid:water (8:2:2). Visualization done by spraying with anisaldehyde sulfuric acid reagent. AF, active fraction; SF-1, SF-
2, SR-3, sub-fractions; C1, compound 1 (lower compound); C2, compound 2 (upper compound). B. HPTLC profile of active fraction showing 10 peaks and compound 1 and 2 from the
AF at 580 nm. The HPTLC was done on precoated aluminum plates of silica gel 60 F254 (Merck). The Rf value of compound 1 was 0.09 and that of compound 2 was 0.69. AU, arbitrary
units.
 R. Bino Kingsley et al. / Journal of Young Pharmacists 5 (2013) 7e12 11

13
Table 3 C NMR (DMSOed6 400 MHz): d 129.60 (C,Ce1&4) 125 (C,Ce2&3),
Effect of chemical fractions and principles isolated from the active fraction on 115 (C,Ce9&10), 79.25 (C,Ce6), 73.13 (C,Ce7), 69.93 (C,Ce5&8),
glucose tolerance in fed and glucose loaded normal rats.
64.87(C,C2&3eOCH3), 58.69 (C,C5eOCH3), 56.58(C,C8eOCH3), 56.29
Serum glucose levels (mmol/L) (C,Ce13), 50.40 (C,Ce12), and 15.17(C,Ce14&15) ppm.
Treatment Time (min) after glucose administration Based on the spectra the second active principle, compound 2
(C2) was a naphthoquinone derivative, 5,8-dihydro-7-isopentyl-
0 (initial) 30 90
2,3,5,8-tetramethoxynaphthalene-1,4,6-triol. The structure of this
Control 5.80  0.24 7.81  0.59 6.77  0.30
Active fraction (25 mg/kg) 5.62  0.31 6.47  0.56** 5.85  0.36*
active principle (C2) is shown in Fig. 4.
Sub-fraction-1 (25 mg/kg) 5.96  0.24 6.07  0.46** 5.44  0.26* The scanned spectra of compound 1 and 2 are given as
Sub-fraction-2 (25 mg/kg) 5.85  0.29 8.16  0.25 6.95  0.25 Supplementary information (Fig. 5 and 6).
Sub-fraction-3 (25 mg/kg) 5.99  0.26 6.13  0.27** 5.39  0.19*
Compound 1(2 mg/kg) 5.64  0.29 6.21  0.26** 5.63  0.34*
Compound 1 (5 mg/kg) 5.63  0.24 6.04  0.37** 5.50  0.26* 4. Discussion
Compound 2 (2 mg/kg) 5.66  0.34 6.40  0.40* 5.88  0.29
Compound 2 (5 mg/kg) 5.74  0.29 6.18  0.05** 5.58  0.24*
The iridoid glycoside isolated in the present study is a new
Values are mean  SD, n ¼ 6 in all groups except in control where n ¼ 12; *P < 0.05, molecule, not known previously. The other active compound was a
**P < 0.001(Compared to control); 0 min, initial glucose level just before drug derivative of naphthoquinone, 5,8-dihydro-7-isopentyl-2,3,5,8-
administration; 30 min and 90 min, time after glucose loading [GTT for sub-fractions
and the isolated compound were done separately. Since the control values for the 2
tetramethoxynaphthalene-1,4,6-triol. This molecule is also a new
sets of experiments did not differ significantly they were combined]. molecule, not reported previously. The existence of other minor
active principles in the AF cannot be ruled out. It is of interest to
note that the novel isolated compounds showed in vivo anti-
OCH3), 3.73 (s, 3H,C0 5eOCH3), 3.67 (s, 2H, C7eCH2eOH) and 3.64 hyperglycemic activity at a very low concentration (2 mg/kg).
(d, 2H, C0 6eCH2eOH) ppm. The herbal drug is effective in orally glucose loaded, fed rats, but
13
C NMR (CD3OD 400 MHz): d 151.90 (C, C4eCOOH) 138.56 (C,Ce not in fasted rats.7 The herbal drug showed anti-hyperglycemic
3), 112.37(C,Ce1), 98.67 (C,Ce6),97.87(C,Ce7), 97.00 (C,Ce20 ), 81.87 effect, not hypoglycemic activity.
(C,Ce7), 76.83(C,Ce5), 74.70 (C, Ce40 ), 70.99(C,Ce50 ),70.43 (C, Ce Therefore, in fed rats the herbal drug (AF) may be stimulating
4a), 69.80(C, Ce30 ) 69.01(C,Ce60 ) 68.81(C,Ce7a) 64.43(C,C0 3e the release of one or more known or unknown factors from the
OCH3),63.13(C,C0 4eOCH3), 61.25 (C,C0 5eOCH3), 29.34(C7eCH2eOH) gastro-intestinal tract which may block absorption of glucose
and 22.69 (C,C0 6 eCH2eOH) ppm. directly or indirectly from intestinal tract. This likely mechanism of
1
H NMR (D20, 400 MHz): d 4.14 (s, 1H, C0 6eCH2eOH), 4.11 (s, 1H, action remains to be studied.
C7eCH2eOH), 3.97e3.96 (d, 1H, C5eOH), 3.93 (s, 1H, C0 3eH) and The herbal drug is likely to be useful as an anti-DM medicine. It
3.90e3.88 (brd.1H, C0 5eH). appears to be non-toxic. In the traditional medicine, there is no re-
The molecular formula of C1 was deduced as C19 H28 O11 by the ported or known toxicity to the herbal drug. Further, in the pre-
analysis of the spectra. C1 was an iridoid glycoside, 1,4a,5,7a-tet- liminary toxicity evaluation in rats, the active fraction did not exhibit
rahydro-5-hydroxy-7-(hydroxymethyl)-1-(tetrahydro-6-(hydrox- any toxicity.7 However, toxicity to long time treatment of the active
ymethyl)-3,4,5-trimethoxy-2H-pyran-2-yloxy)cyclopenta[c]pyran- fraction remains to be studied. Although there are many drugs to
4-carboxylic acid. The structure of this active principle (C1) is treat type-2 diabetes in the market, in the long term treatment, im-
shown in Fig. 4. mune response dependent or other toxicity develops; the drug be-
Compound 2 (C2): IR (KBr): 3400 (OeH str.), 2380 (CeH ofe comes ineffective in some cases. In this context, this herbal drug is
CH2e str.), 1600 (CeH (aromatic) str.) and 1250e1300 (CeOeC str.) likely to be useful as an alternative medicine or as a combination
cm1. MS: m/z 371. (Mþ. 368 þ 3) of C19 H28 O7. drug. There is a need to carry on further studies leading to likely
1
H NMR (DMSOed6 400 MHz). d 7.46 (s, 1 H, C1eOH), 7.40 (s, 1 H, development of the active fraction as a standardized, safe and
C4eOH), 7.35e7.32 (d, 1H, C6eOH), 6.89e6.81 (m, 1H,C5eH), 6.75e effective phytomedicine. The active compounds are promising mol-
6.57 (m, 1H, C8eH) 3.73 (s, 6H, C2 e& C3eOCH3), 3.69 (s, 3H, C5e ecules for the development of conventional chemical entity drugs.
OCH3) 3.67 (s, 3H, C8eOCH3), 3.54e3.29 (m, 5H, C11,12&13eH) and In conclusion, the active fraction (AF) from S. tetragonum DC.
15.17 (d, 6H, C13eCH3)ppm. showed anti-diabetes activity against streptozotocin-induced type-

Fig. 4. Structures of compounds 1 and 2.

12 R. Bino Kingsley et al. / Journal of Young Pharmacists 5 (2013) 7e12
2 diabetic rats. In contrast to oral glucose administration, when
glucose was loaded intra-peritoneally to normal rats, the AF did not
exhibit anti-hyperglycemic activity in glucose tolerance test. The AF
did not significantly influence insulin release from cultured rat
pancreatic islets. The major mechanism of action could be indirect
inhibition of glucose absorption or transport from intestine. Two
active principles (active at 2 mg/kg body weight) were isolated and
characterized with spectral data. These active molecules are very
promising for further studies leading to the development of a
valuable medicine for mono-therapy and/or combination therapy
for diabetes.
Conflicts of interest
All authos have none to declare.
Acknowledgment
Dr. B. Sabulal, Head, Phytochemistry and Phytopharmacology
Division, TBGRI, and Dr. Ajikumaran Nair and Dr. Anil John, Tech-
nical Officers, TBGRI are acknowledged for their help in the
experiments
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.jyp.2012.09.002.
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